Copyright 0 1993 by the Genetics Society of America

Mapping Chromosome Rearrangement Breakpoints to the Physical Map of Caenorhabditis elegans by Fluorescent in Situ Hybridization

Donna G. Albertson

Laboratory of Molecular Biology, Medical Research Council, Cambridge GB2 2QH, England Manuscript received November 15, 1992

Accepted for publication January 22, 1993

ABSTRACT A scheme for rapidly mapping chromosome rearrangements relative to the physical map of

Caenorhabditis elegans is described that is based on hybridization patterns of cloned DNA on meiotic nuclei, as visualized by fluorescent in situ hybridization. From the nearly complete physical map, DNA clones, in yeast artificial chromosomes (YACs), spanning the rearrangement breakpoint were selected. The purified YAC DNAs were first amplified by degenerate oligonucleotide-primed polymerase chain reaction, then reamplified to incorporate fluorescein dUTP or rhodamine dUTP. The site of hybridization was visualized directly (without the use of antibodies) on meiotic bivalents. This allows chromosome rearrangements to be mapped readily if the duplicated, deficient or translocated regions do not pair with a normal homologous region, because the site or sites of hybridization of the probe on meiotic prophase nuclei will be spatially distinct. The pattern, or number, of hybridization signals from probes from within, or adjacent to, the rearranged region of the genome can be predicted from the genetic constitution of the strain. Characterization of the physical extent of the genetically mapped rearrangements places genetic landmarks on the physical map, and so provides linkage between the two types of map.

T HE success of combining genetic and molecular approaches to the study of the normal and ab-

normal biology of an organism has highlighted the need for a physical map of the organism’s genome and ultimately its entire DNA sequence. For a number of organisms cytological and genetic maps exist along with partial or complete physical maps, and from comparison of these maps, it is clear that the propor- tion relating genetic and physical (or cytogenetic) maps is not constant across the genome (LINDSLEY and SANDLER 1977; DONIS-KELLER et al. 1987; COUL- SON et al. 1991). In general, it appears that the distal portions of autosomes are regions where recombina- tion is frequent, leading to genetic map expansion between distal loci. Therefore, for the physical map to be fully utilized, it is necessary to establish the correspondence between the genetic and physical maps across the entire genome. Linking of the two types of map can be accomplished by molecularly cloning genetically characterized genes, by identifying mutations in physically mapped genes, by identifying genetically informative DNA polymorphisms, or by the physical and genetic characterization of chromo- somal rearrangements or breakpoints.

In the nematode Caenorhabditis elegans, the nearly complete physical map (COULSON et al. 199 1) has been tied to the genetic map by molecular cloning of ge- netically identified genes. This has involved direct transposon tagging of a gene (GREENWALD 1985) or mapping of closely linked DNA polymorphisms (BAIL- LIE and BECKENBACH 1985). Genetic mapping of DNA

Genetics 134: 21 1-219 (May, 1993)

polymorphisms has also contributed genetic and phys- ical landmarks on the maps (HIRSH et al. 1979; ROSE et al. 1982; FILES, CARR and HIRSH 1983), and re- cently, a genetic mapping system involving sequence- tagged sites has been described (WILLIAMS et al. 1992) that not only provided genetic landmarks on the phys- ical map, but also contributed to the construction of the map by locating previously unassigned DNA con- tigs.

T h e chromosomes of C. elegans are holocentric (ALBERTSON and THOMSON 1982) and therefore chro- mosome rearrangements involving translocations, de- ficiencies, o r small pieces of chromosomes, called free duplications, are readily isolated and maintained [for a review, see HERMAN and KARI (1 989)]. These also provide a source of genetic markers delimiting regions of the genome that can link the physical and genetic maps, but only limited use has been made of chro- mosome rearrangements for this purpose. Using cy- togenetics, DNA clones have been mapped relative to chromosome rearrangement breakpoints in order to identify corresponding genetic loci (ALBERTSON 1985) and, for the cloning of particular genes, the extents of small deficiencies (KRAMER et al. 1988) or duplica- tions (H. BROWNING, personal communication) have been mapped relative to the physical map.

Since genes are often mapped relative to re- arrangements, it seemed appropriate to map a number of the rearrangements with respect to the physical map, thereby providing more genetic landmarks on the physical map, and at the same time physical char-

212 D. G. Albertson

acterization of some of the chromosome re- arrangements. One way of doing this is to use the physical map and the available genetic data to select D N A clones mapping near the rearrangement and to map these by in situ hybridization to the rearranged chromosomes. Both breakpoints and the extent of duplicated regions, for example, can be mapped rel- ative to the physical map in this way. However, to map genes cytologically in C. elegans, DNA probes are hybridized in situ to the very few mitotic chromosomes obtained by squashing embryos from large numbers of animals (ALBERTSON 1984a, 1985). Since chromo- some rearrangements are often lethal or unstable, they are maintained as heterozygotes, and therefore metaphase karyotypes of embryos from these mothers will be of several different constitutions and the num- ber of metaphases obtained from squashes too small for analysis of a number of rearrangements. There- fore a different strategy has been adopted. Since a large number of meiotic prophase nuclei can be ob- tained from an individual animal, DNA clones have been mapped to these chromosomes obtained from a few selected individuals of a specific genotype. Al- though the meiotic bivalents are so small that the position of hybridization along the chromosome can only be determined at very low resolution, it is only necessary to score for the presence or absence of hybridization signals on a specific number of bivalents depending on the particular genotype. The analysis makes use of the existing resource of purified genomic DNA cloned in yeast artificial chromosomes (YACs) used to construct the physical map (COULSON et al. 1988). Since in situ hybridization typically requires as much as 0.1 pg of DNA per slide (ALBERTSON 1984), the isolated YACs, stored as gel slices, were amplified by the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) (TELENIUS et al. 1992a,b) and then reamplifed incorporating directly fluores- cently labeled deoxynucleotides for use as probes for in situ hybridization. These YACs, labeled by DOP- PCR, were mapped on metaphase chromosomes, dem- onstrating that DOP-PCR can be used to generate probes for in situ hybridization in organisms lacking the interspersed repeats often used to prime labeling by techniques such as Alu-PCR (BREEN et al. 1992; LENGAUER, GREEN and CREMER 1992). The large hybridization signals from these directly fluorescently labeled YAC probes renders characterization of chro- mosome rearrangements relatively easy in an orga- nism with otherwise “difficult” cytogenetics.

MATERIALS AND METHODS

Nematode strains: The strains used were all derived from C. elegans var. Bristol and maintained as described by BREN- NER 1974). The strain CB1517 (HODGKIN 1980), with the genetic constitution eDj2/eDf2 Ill;eDp6(lll;f) was obtained from J. HODGKIN. The mnDp33(X;IV) rearrangement (HER- MAN, MADL and KARI 1979) in strain SP309 (genetic consti-

tution: mnDp33/+ ZV; unc-ZO(el12) X ) was obtained from R. K. HERMAN and the mnTZ(X;ZI) translocation (HERMAN, KARI and HARTMAN 1982) in strain SP400 (genetic consti- tution: mnTl l (X; l I ) /+ Il;dpy-3(e27) X;mnDpl l (X; l l ;J ) was obtained from the Caenorhabditis Genetics Center.

Labeling YACs by DOP-PCR. Purified YAC DNAs (COULSON et al. 1988) were heated at 65’ and a 1-wl aliquot was taken for amplification by DOP-PCR as described by TELENIUS et al. (1 992b). Theoretically, priming is predicted at every 4 kb (TELENIUS et al. 1992a) and the amplified products, when electrophoresed through short agarose gels typically displayed more than 12 bands. A 5-pI aliquot was subjected to a second amplification of 30 cycles, omitting the low temperature annealing cycles used in the first am- plification. The amplified YAC DNA was labeled in the second reaction by the addition of 30 PM fluorescein dUTP (Fluorescein-1 1-dUTP, Amersham; Fluorescein-1 2-dUTP, Boerhinger-Mannheim) or rhodamine-4-dUTP (FluoroRed, Amersham) and reduction of the dTTP concentration to 160 PM. The labeled product was used for in situ hybridi- zation without purification by adding 0.5 P I of the PCR product to 20 PI of hybridization solution consisting of 50% formamide, 0.01 M piperazine-N,N’-bis[2-ethane-sulfonic acid] (PIPES), 0.001 M EDTA, pH 7.0, 10% dextran sulfate and 0.3 M NaCl (ALBERTSON 1984a).

Cytology: Individual animals were picked and cut to release the gonad, as described previously (ALBERTSON 1984b), then fixed in ethano1:acetic acid 3:l for 30-60 min before processing for in situ hybridization as described pre- viously for embryonic squashes (ALBERTSON 1984a).

Hybridization: The probe (20 P I ) was denatured, as de- scribed previously (ALBERTSON I984a) and then applied to the slide and covered with a 12 x 12-mm piece of Parafilm. Slides were incubated at 37’ for 1-1 6 hr in a moist chamber, then washed as described previously (ALBERTSON 1984a). For visualization of rhodamine labeled YACs, the chromo- somes were stained with 0.3 rg/ml 4’,6-diamidino-2-phen- ylindole (DAPI) and for visualization of fluorescein-labeled YACs, with 100 wg/ml propidium iodide. The slides were rinsed briefly in 10 mM Tris, pH 9.5 and mounted in 1,4- dia7.obicyclo[2,2,2]octane (DABCO) to reduce fluorescence fading (JOHNSON et al. 1982).

Microscopy: The hybridization signals from fluorescein- labeled YACs on propidium iodide-stained chromosomes were visualized using a Bio-Rad MRCGOO confocal micro- scope, as described previously (ALBERTSON, SHERRINGTON and VAUDIN 199 1). The site of hybridization of rhodamine- labeled YACs was visualized using conventional fluorescence microscopy and an intensified CCD camera, as described previously (ALBERTSON, SHERRINCTON and VAUDIN 199 l ) , using Zeiss filter sets 00 and 2.

RESULTS

Mapping YAGs on embryonic metaphase chro- mosomes: In the absence of interspersed repeats to specifically prime labeling of the genomic DNA in- serted in the YAC, it was necessary to use purified YAC DNA for the generation of DNA probes. A source of purified YACs was available, as these had been produced in the course of construction of the physical map and stored as frozen gel slices (COULSON et al. 1988). In order to conserve this resource, a 1-p1 aliquot of the slice was amplified by DOP-PCR (TE- LENIUS et al. 1992a,b). A second amplification reaction was carried out to incorporate either fluorescein dUTP or rhodamine dUTP. This labeled DNA was

Mapping Chromosome Rearrangements 213

distance from the genetic left end of the chromosome (ALBERTSON 1985). As expected, the YAC hybridized to the left end of chromosome I, identified by the hybridization of a ribosomal DNA probe to the right end of linkage group I (ALBERTSON 1984a). A second, unmapped YAC, Y40B6, was then mapped to the middle of linkage group I (Figure lb). The position of this YAC on the physical map of chromosome I was independently assigned by physical methods (A. COUL- SON and R. SHOWNKEEN, personal communication). The distribution of the hybridization signals from the YACs along metaphase chromosomes is comparable to that obtained when using cosmids. Thus, these YACs, directly, fluorescently labeled by DOP-PCR, are suitable probes for in situ hybridization on both chromosomes and interphase nuclei (Figure IC).

Mapping chromosome rearrangements: Mapping of genes to C. elegans chromosomes by cytogenetics has generally involved the assignment of hybridization signals to positions on embryonic metaphase chro- mosomes identified by a second marker, as described above. The use of these metaphase chromosomes to map chromosome rearrangements presents several problems, since few chromosomes are obtained in this way and most rearrangements are maintained as het- erozygotes so that embryonic metaphases will have different karyotypes. A more uniform and abundant supply of chromosomes is available however in meiotic prophase nuclei. In adult hermaphrodites, meiotic stages are linearly disposed along each of two arms of

FIGURE 1 .-Hybridization of fluorescein dUTP-labeled YACs to the gonad with many pachytene nuclei in the distal

embryonic metaphase chromosomes. Hybridization of fluorescein portion of the gonad in which chromosomes are visible dUTp-]abeled Y A C ~ and the ribosomal DNA probe tO embryo but not usually individually distinct. Proximally, the squashes from wild-type animals was visualized by confocal micros- nuclei arrest in diakinesis prior to fertilization and, copy. The microscope was operated in the dual channel mode, the typically, in three or mOre nuclei, depending on age images from the two channels were merged and differentiated in false color (red, propidium iodide; green, fluorescein). (a) Hybridi- and genotype, the meiotic bivalents can be distin- zation of Y75A4 (arrowheads) and the ribosomal probe (larger guished- The pattern of hybridization to meiotic biv- signals) to opposite ends of linkage group I . There are approxi- alents in these nuclei can be predicted from the gen- mately 100 copies of the 7-kb ribosomal DNA repeat, and the size otype of the animal , whi]e confirmation of a duplica- of the YAC, Y75A4, from mobility on pulsed field gels is estimated to be 100 kb. Therefore, the genomic target size of the YAC probe tion’ for example9 might be Obtained from Observation is approximately one-seventh that of the ribosomal probe. (b) Hy- of multiple hybridization signals Over the early pro- bridization of Y40B6 (arrowheads) and the ribosomal probe (larger phase nuclei where individual bivalents are not visible. signals) to linkage group 1. ( c ) Hybridization of Y40B6 (smaller Since each animal contains a number of nuclei suitable signals) and the ribosomal probe (larger signals) to interphase nuclei from embryo squashes. Scale bar is 10 pm. for mapping by in situ hybridization, only a few ani-

mals are required. For each YAC to be mapped with shown to be a suitable probe for in situ hybridization by mapping a YAC, Y75A4, already positioned on the left end of the physical map of linkage group I . Figure la shows the hybridization of the YAC and the ribo- somal DNA probe to a metaphase spread from a wild- type C. elegans embryo. Since the chromosomes are indistinguishable, either morphologically distinct re- arrangements or a second gene probe (in this case the ribosomal genes) is used to label the chromosome, and the site of hybridization is assigned as the percentage

respect to a rearrangement, approximately five ani- mals of the desired genotype were picked to a glass microscope slide and processed for in situ hybridiza- tion. In order to determine the physical extents of the rearrangements, the number of fluorescent hybridi- zation signals on pachytene nuclei and the pattern of hybridization signals on meiotic bivalents (univalents, in the case of a single X chromosome) were scored.

The eDf2 and eDp6 breakpoint: Due to the holocen- tric nature of C. elegans chromosomes, breakage of a

214 D. G . Albertson

Meiotic Karyotype Hybridization to

Chromosomes a. eDfZ;eDp6 I I1 8Df2(111) IV V X 8Dp6 e012 eDp6

888888888888 OD

88888888888 a.

888888888888

+ -

+ +

- +

b. IV/mnDp33 (X;Iv) I I1 111 IV/mnDp33 V X mnDp33 X

888888 88 888 - t

888888 88 888 + +

c. mnTl l(Y;II)/+lI; dpy-3(X); mnDpl l(X;ll;f) X-Linked YACS

I Il/mnTll 111 IV v x mnDp11 mnDp11 mnTl1 X / I

8 8 & - j 8 8 8 8 8 S c b a. + - + -

88#88888816 + + + -

Linkage group 11 YACS - + - +

+ - - +

FIGURE 2.-Meiotic karyotype at prophase I and hybridization pattern of YACs relative to rearrangement breakpoints. Idiograms of meiotic chromosome constitutions of strains carrying chromo- some rearrangements. Since the chromosomes are holocentric, centromeres are absent and the homologs are held together in an end-to-end association, possibly through terminalized chiasmata. Either genetic end of the homologs can be involved in the end-to- end association, such that hybridization of a probe from one end of a chromosome can be observed at the inside or the outside of the bivalent in different nuclei (ALBERTSON and THOMSON 1993). Therefore for a probe mapping near the end of a chromosome, some bivalents will be oriented such that hybridization of the probe is to the outside of the bivalent and in animals heterozygous for that probe hybridization to only one half bivalent will be observed as in Figure 7. Although all four chromatids have been drawn, they are rarely distinguishable in these squash preparations. However, a constriction is usually visible, that indicates the site of association of the two half bivalents. The pattern of hybridization of YACs to meiotic bivalents (filled) has been drawn. The inclusion (+), or absence (-) of the YAC on a chromosome is indicated by these hybridization patterns. The number of hybridization signals over early meiotic prophase nuclei will be determined by the number of different bivalents to which the YAC hybridizes. (a) eDfl;eDp6. Linkage group I 1 1 is rearranged such that the eDfl-bearing chro- mosome appears as a normal bivalent. The eDp6 chromosome is easily distinguished from the six bivalents by its small size. (b) mnDp33(X;IV). Animals heterozygous for mnDp33 display six biva- lents in meiotic prophase I . Since the duplicated portion of the X has been translocated to linkage group IV, this bivalent is composed of one normal linkage group IV half bivalent and the linkage group IV half bivalent carrying mnDP33. (c) mnT2(X;II).

chromosome followed by recovery of both products is possible, as acentric fragments are rarely produced. Breakage of linkage group 111 to generate a chromo- some deficient for approximately 50% of the chro- mosome and recovery of the remainder as a free duplication in one animal has been described and well characterized genetically (HODGKIN 1980). These an- imals are viable and their meiotic chromosome consti- tution is drawn in Figure 2a. Of the six linkage groups of C. elegans, only the third linkage group is altered in this strain. Since the majority of linkage group 111 is intact, it appears cytologically as a normal bivalent, referred to here as the eDf2"bearing chromosome. The other small piece of linkage group 111 can exist in addition to normal linkage group 111 chromosomes as a small free chromosomal duplication and so is named eDp6. Thus, rather than the normal comple- ment of six bivalents, meiotic nuclei in diakinesis in these animals contain an additional small chromo- some. As shown in Figure 2a, YACs that map to the left of the breakpoint can be identified by hybridiza- tion to the eDf2-bearing chromosome, while those to the right of the breakpoint should hybridize to the eDP6 chromosome.

Since the breakpoint of eDf2 and eDp6 has been well mapped genetically, only four YACs with minimal overlap were selected and labeled. The YAC Y53F7 covers the genes unc-50 and unc-69, known to map to the left of the breakpoint (Figure S ) , and therefore, as expected, this YAC mapped on the eDf2-bearing chromosome, while the YAC Y39F2, covering pha-l and tra-I, mapping to the right of the breakpoint hybridized to the eDp6 chromosome (Figures 3b and 4c). The hybridization pattern of the remaining two YACs showed that Y40C2 maps to the left of the breakpoint (Figures 3b and 4a) and that since Y59A1 mapped to both the eDf2-bearing chromosome and eDp6 (Figures 3b and 4b), the breakpoint must lie within the genomic DNA cloned in this YAC.

Mapping breakpoints to provide genetic markers on the Zejt end ofthe X: For the leftmost 25% of the current physical map of the X chromosome, only two genetic markers have been linked to the physical map. A number of chromosomal rearrangements involving this region have been described and mapping of two of these is described here.

The duplication mnDp33 is homozygous lethal and includes the X-linked genes osm-5, unc-20, unc-78 and Zin-18 translocated to linkage group IV (HERMAN,

This reciprocal translocation is made up of the two half transloca- tions mnTl I(X;II) and mnDpl l(X;II;J. Since the mnT1 I half trans- location pairs with and disjoins from a normal linkage group I I chromosome, this bivalent is composed of the normal linkage group I 1 half bivalent and the slightly longer appearing mnTI I half biva- lent. A single X chromosome is present in these animals and this univalent is morphologically distinct. The mnDp1 I half transloca- tion can also be distinguished as a small chromosome.

Mapping Chromosome Rearrangements 215

FIGURE 3.-The breakpoint of eDj2 and eDp6. (a) T h e genetic map of linkage group 111 with the genetic left end up and showing genetic markers mapping close to the breakpoint. (b) A physical map of linkage group 111. This physical map representation of linkage group 111 was redrawn from the “Physical Chromo Map” display in the C. eleguns database ACEDB (R. DURRIN and J. THIERRY-MIEG, personal communication). T h e contiguous regions of the physical map are shown as hatched boxes, with arbitrary small gaps between contigs. Two scales are shown; one indicates the percentage length along the chromosome from the left end and the other scale, in megabases, is calculated from the number of Hind111 bands after fingerprinting clones (COULSON et al. 1986). The YACs mapping to the eDf2-bearing chromosome (B) or to the eDp6 chromosome (0) are shown to the right of the physical map at their physical map locations. The YAC Y39Al @) mapped to both the eDf2-bearing chromosome and eDp6. Several molecularly cloned genes, mapping near the breakpoint, are shown to the left of the physical map.

MADL and KARI 1979). For mapping, animals of gen- otype mnDp??/+;unc-20, which are phenotypically wild type hermaphrodites, were picked and processed for hybridization with a set of YACs from the left end of the X chromosome. Since few genetic and physical markers were available to select the set of Y A G , the YACs were chosen based on previous in situ hybridi- zation mapping of cosmids covering the X-linked genes vit-?,4, gpd-a,?, vit-5 and vit-2 (see Figure 5b) to mnDp??(X;ZV) chromosomes along with a cosmid

FIGURE 4,”Hybridization of YACs to bivalents from eDf2;eDp6 hermaphrodites. The site of hybridization of rhodamine dUTP- labeled YACs (red false color) was visualized on DAPI-stained chromosomes (blue false color). The separate images from the two fluorochromes were aligned, then merged and displayed in false color. (a) Hybridization of Y40C2 to one bivalent identified as the eDf2-bearing chromosome. The eDp6 chromosome is easily distin- guished from the bivalents by its small size and has not hybridized. (b) Hybridization of the YAC Y39A1 to a meiotic bivalent (eDf2) and eDp6. (c) The YAC Y39F2 hybridized to the eDp6 chromosome. No signal is observed on any of the six bivalents.

containing unc-22(ZV). All these genes, except vit-2 were found to be linked to unc-22 on mnDp?? chro- mosomes (data not shown). Therefore the right break- point of mnDp?? was located between vit-2 and vit- ?,4. Figure 2b shows the meiotic karyotype of these animals. T h e duplication, carried on one linkage group IV chromosome is too small to visibly alter the chromosome cytologically. Therefore these animals appear to have six normal bivalents. Inclusion of a

D. G . Albertson 216

a.

mnDp33

0 5 map units

FIGURE 5.”The region of the X duplicated by mnDp33(X;IV). (a) The X-linked genetic markers included in mnDp33. (b) Physical map of the X chromosome, drawn as in Figure 3. The YACs included 0 in mnDp33, or tested. but not included (0) are shown to the right of the physical map. The positions of some molecularly cloned genes are shown to the left of the physical map, together with two sequence-tagged sites, srP4O and srP41.

YAC in the duplicated portion of the genome was indicated by hybridization of the YAC to two sites in pachytene nuclei, or to two bivalents at diakinesis. The assignment was confirmed by observing “sym- metric” hybridization of the X-linked YAC to the X meiotic bivalent, but “asymmetric” hybridization, that is, to only the mnDp33 half bivalent of linkage group IV. The YACs included in m n D p 3 3 are shown i n Figure .5.

The reciprocal translocation, m n T 2 (HERMAN, KARI and HARTMAN 1982) is composed of the two half translocations called m n T l l(X;II) and m n D p l I(II;X;J, as shown in Figure 6. The meiotic karyotype of the animals selected for mapping is shown in Figure 2c. Phenotypically wild type hermaphrodites carried, in addition to normal linkage groups I , 111, IV and V, one normal linkage group I 1 chromosome that pairs with and disjoins from mnTI I , a single X chromosome and the free duplication mnDp1 I. As shown in Figure

2c. the X-linked breakpoint of the translocation was determined by mapping X-linked YACs to these ani- mals and looking for hybridization signal not only on the univalent X, but also, on either m n D p l I or asym- metrically to the m n T l I half bivalent. Similarly, the breakpoint on linkage group 11 was determined from hybridization of YACs symmetrically to the linkage group 11 and m n T l I half bivalents or asymmetrically to the normal linkage group I 1 half bivalent and mnDp1 I (Figures 2c and 7 ) . The results of these hybridizations are shown i n Figure 6. The X-linked breakpoint falls between Y8D1 and Y42C9, while the breakpoint on linkage group I 1 is within the region of genomic DNA cloned i n Y70H7, since this YAC hy- bridized to both m n T l I and m n D p l 1 , as well a s to the normal linkage group I I chromosome.

DISCUSSION

A scheme for rapidly mapping chromosome re- arrangements relative to the physical map of C. elegans has been described that is based on hybridization patterns of YACs on meiotic nuclei. From the nearly complete physical map (COULSON et al. 1991). YACs potentially covering ;I rearrangement breakpoint can be predicted, selected and then labeled for in situ hvbridization. I n the absence of interspersed repeats to prime labeling, DOP-PCR was used first to amplify, then incorporate directly fluorescentlv labeled nucle- otides into purified YACs. This PCR product was suitable for in situ hybridization without purification and hybridization times as short a s 60 min resulted i n visible hybridization signals. The analysis was speeded up further by direct visualization of the fluorescence from the rhodamine or fluorescein molecule coupled to the dUTP incorporated i n the probe DNA. This not only saved time i n processing samples, because antibody incubations are omitted, but also allows the use of cytological material that is apt to give significant background fluorescence from antibody binding. The many meiotic nuclei i n the gonad of the organism provide a source of large numbers of chromosomes, difficult to obtain from asynchronous populations of embryos. The hybridization pattern on the meiotic nuclei from a s few ;IS five animals can indicate the location of the YAC relative to the chromosomal rearrangement breakpoint. Since the aim of this work was to develop a means of mapping ;I number of rearrangements, YACs, and often the larger YACs, were chosen as probes i n order to cover ;I chromo- somal region efficiently. However, the same labeling and mapping strategy can be used with cosmids as probes, although this is slightly more difficult because smaller probes are used.

I n some cases YACs spanning the breakpoint could be identified when hybridization signals on both halves of a translocation, for example, were observed (Figure 7 ) . Although the breakpoints have not been

Mapping Chromosome Rearrangements 217

a. b. X

0%

10% I

100% :II

11

Y 4 7 U 0% I Y51EZ

'm Y29F10 ,0% - Y42C9

~ Y8D1

20%

30%

40%

50%

60%

70%

em

90?4

1 00%

FIGURE 6.-The breakpoints of mnT2(X;II) on linkage groups II and X . (a) Genetic map of the breakpoints of the two half translocations, m n T l 1 and mnDpl1 . (b) The physical map of the X and chromosome II drawn as in Figure 3. The YACs included in m n D p l l o or m n T I l @ ) are shown to the right of the physical maps of the two chromosomes. The YAC, Y70H7 (0) from chromosome II mapped to both m n T l l and mnDpI1. Several genetic markers that have been molecularly cloned are shown to the left of the physical map.

Y49F8

165C11

FIGURE 7.-Hybridization of Y65C11 to one half bivalent and to m n D p l l (arrows). The site of hybridization of the rhodamine labeled YAC (red) was visualized on DAPI-stained chromosomes (blue) displayed here in false color after aligning and merging the two separate images of the two different fluorochromes. The two hybridization signals, one on each chromatid, identify this half bivalent as linkage group 11. The failure of the YAC to hybridize to both half bivalents indicates that Y65C11 is not included on the portion of linkage group I1 carried on mnT11, but is included on mnDpl1 , as seen by the hybridization signal on this small chromo- some.

localized to cosmids, it appears from these YACs that reliable hybridization signals can be seen when half a YAC (approximately 100 kb) is included on the rear- ranged chromosome. However, it is also likely that the nature of the breakpoint itself will greatly influ- ence the hybridization characteristics of the YACs, since in the formation of the rearrangement small duplications, deletions or inversions may have oc- curred locally.

The extent of deficiencies can also be mapped cy- togenetically in this way, although not reported here. I t would be necessary to score more animals if the

deficiency were maintained in a heterozygous strain, since the mapping would require distinguishing asym- metric hybridization to bivalents at diakinesis. Obser- vations of asymmetric hybridization to the mnDp33, mnTl I or linkage group I1 (Figure 'I) half bivalents are examples of this type of analysis.

Since genes are often genetically mapped relative to chromosome rearrangements, the assignment of breakpoints to the physical map should facilitate the molecular cloning of genes. In some instances, chro- mosome rearrangements will serve as markers to de- limit a region of the physical map within which a particular genetic locus will be found. In a few cases, breakpoints might identify a gene. This appears to be true for the breakpoint of eDj2 and eDp6. Although homozygous-viable, the eDf2;eDpb animals are un- coordinated. A gene, unc-119, with a similar pheno- type has been mapped very close to, or within, the breakpoint, since this gene is neither included in eDj2, nor is it covered by eDp6 (D. PILGRIM and J. HODGKIN, personal communication). Location of the breakpoint within Y39A1 should facilitate the molecular cloning of this gene.

The breakpoint of eDj2 and eDp6 had been quite well mapped genetically and only four YACs were required to cover genetic markers mapping to either side of the breakpoint. The linkage group I1 break- point of rnnTZ(X;II) had also been well mapped genet- ically, close to and to the right of sqt-1 (previously called rol-5), by demonstrating that mnDpl l(X;ZZ;j did not carry a functional sqt-1 gene, although it was not possible to demonstrate the presence of a func- tional sqt-1 gene on mnTl1 (HERMAN, KARI and HARTMAN 1982). The breakpoint of mnT2 on linkage

218 D. G. Albertson

group I1 was mapped to Y70H7, since this YAC mapped to both m n T l I and mnDpl I. The genetic location of the breakpoint near sqt-1 is confirmed, since Y70H7 covers sqt-I. Also consistent with these genetic data were the observations that the YACs (Y49F8 and Y62F5) physically mapped near sqt-I and to the right of sqt-1 both hybridized to mnDpl I , while the YACs, Y50G2 and Y54E11, physically mapped to the left of sqt-1 hybridized to m n T I I . However, an additional YAC covering sqt-I (Y8E4) mapped only to mnDpl1. From physical mapping of cDNA clones (WATERSTON et al. 1992) to both Y54E11 and Y70H7, but not Y8E4, it appears that Y70H7 extends further to the left than Y8E4. Therefore the breakpoint is more likely to be near sqt-1 and to the left, rather than to the right of this gene. This apparent contra- diction could be explained if, in the creation of mn- D p l 1 , multiple breaks or rearrangements occurred, that have rendered sqt-1 inactive. Since the initial description of mnT2 (HERMAN, KARI and HARTMAN 1982), several small deficiencies and lethal mutations have been isolated and mapped to this region (SIG- URDSON, SPANIER and HERMAN 1984) and genetic complementation tests between some of these and mnDpl1 might help to characterize the breakpoint.

Aside from characterizing the rearrangements themselves, the mapping of rearrangement break- points places genetic landmarks on the physical map. The left end of the X chromosome is one area of the genome where few genetic markers have been placed on the physical map, yet genetic studies suggest that analysis of DNA sequences in this region would be interesting. For example, it is within this region that DNA sequences likely to be important for the homol- ogous pairing of the X chromosome in meiosis are thought to reside (HERMAN and KARI 1989). Previ- ously, two sequence-tagged sites were mapped to the left end of the X (WILLIAMS et al. 1992). Now, by mapping the breakpoints of the two chromosomal rearrangements, mnT2 and mnDp33, as reported here, three more landmarks have been added to this region of the physical map. From the physical map it is possible to locate the breakpoint of mnT2 on X , pre- viously mapped between unc-1 and dpy-3, to the inter- val between stP4I and dpy-3, since Y8D1, which maps to the right of stP4I was included on mnDpl1 (Figure 6b). For the region covered by mnDp33, the location of stP40 in Y60B6 provides a physical and genetic marker to subdivide the duplicated region. In addition to the genes genetically mapped to the region covered by mnDp33, a number of genes identified by molecular cloning or by tagged sequencing of cDNAs (WATER- STON et al. 1992) have been mapped to this region of the physical map. Assignment of physical endpoints to mnDp33 should facilitate design of genetic screens for identification of mutations in some of these genes.

The extent of mnDp33 is entirely contained within

a contiguous portion of the physical map. An estimate of the physical size of the duplicated region of the X is therefore possible from the physical map. As shown in Figure 5b, mnDp33 spans approximately 1.75 Mb of DNA and approximately 6 map units on the genetic map. Therefore, for this region of the X chromosome, 1 map unit corresponds to 300 kb. This is similar to an average rate calculated over the genome as a whole, and provides a contrast to the suppression of recom- bination that has been observed on several autosomes, where the ratio of the physical and genetic metrics can be up to 1-2 Mb/map unit (GREENWALD et al. 1987; PRASAD and BAILLIE 1989; STARR et al. 1989). It has been noted previously that the X differs from the autosomes in that it is largely devoid of the genetic clusters that characterize the autosomes (BRENNER

The analysis of chromosome rearrangements de- scribed here relies on prior knowledge of the genetic constitution of the rearrangement. It cannot therefore detect unexpected alterations in the genome outside the region covered by the selected YAGs. Small defi- ciencies or additions, such as telomeric sequences at the ends of free duplications, cannot be identified. In the analysis of translocations in human cell lines, a more complete picture of the composition of a rear- ranged chromosome has been obtained by labeling flow sorted chromosomes (TELENIUS et al. 1992a,b), or microdissected chromosomes (MELTZER et al. 1992) and “painting” these back onto normal human meta- phases by in situ hybridization. In this way unexpected sequences contained in the rearrangement may be visualized. In plants and animals where somatic met- aphase chromosomes may be difficult to obtain in large numbers, these methods for characterizing chro- mosomal rearrangements may be less useful, since microdissection and flow sorting may be difficult. In C. elegans, as in many other organisms, when meta- phase chromosomes are obtained they are small, and identification of the chromosomes in normal meta- phases requires a set of in situ hybridization markers in addition to the specific chromosomal painting probes. Often meiotic tissues provide a richer source of chromosomes, and use of these chromosomes for in situ hybridization mapping (MOENS and PEARLMAN 1990; ALBINI and SCHWARZACHER 1992) appears to be a good alternative to metaphases, in some cases allowing high resolution cytogenetic mapping. Even at relatively low resolution, cytogenetics offers an efficient method for mapping chromosome re- arrangements to unite physical and genetic maps.

1974).

The purified YAC DNA, as gel slices, was kindly provided by JOHN SULSTON, ALAN COULSON and RATNA SHOWNKEEN. I thank H. TELENIUS for sharing his unpublished protocols for DOP-PCR and RICHARD DURBIN for help with the C. eleguns database ACEDB. Some strains were provided by the Caenorhabditis Genetics Center. This work was carried out, with support from the Medical Research Council, as part of the UK Human Genome Mapping Project.

Mapping Chromosome Rearrangements 219

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Communicating editor: R. K. HERMAN